Compounds that abrogate DNA damage induced cell cycle G2 checkpoint and/or augment anti-cancer activity of DNA-damaging treatments

ABSTRACT

The invention provides compositions and methods to inhibit the cell cycle G2 checkpoint, in particular the DNA-damage-induced G2 checkpoint, in mammalian cells including human cells. Specifically, the invention provides compositions and methods to sensitize cells to DNA-damaging agents by abrogating the cell cycle G2 checkpoint. Compounds of the invention are used to treat proliferative disorders such as cancer. The invention provides compositions and methods for selectively sensitizing G1 checkpoint impaired cancer cells to DNA-damaging agents and treatments.

RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.60/386,930, filed Jun. 6, 2002.

FIELD OF THE INVENTION

The present invention relates to chemical compounds having anti-cellproliferative activity, and to their production, as well as topharmaceutical compositions containing them, and to methods of treatingproliferative disorders using these compounds and compositions. Theinvention compounds are therefore useful for inhibiting cellproliferation and, as such, for treating cell proliferative disordersincluding cancer. In particular, the invention relates to compounds thatabrogate the cell cycle G2 checkpoint, including the DNA-damage-inducedG2 checkpoint, which are useful in treating proliferative disorders suchas cancer, including treating metastatic and non-metastatic solid orliquid tumors.

BACKGROUND

The cell cycle comprises S phase (DNA replication), M phase (mitosis),and two gap phases (G1 and G2 phases) between S and M phases.Checkpoints in the cell cycle ensure accurate progression through cellcycle stages, and include monitoring the state of DNA integrity, DNAreplication, cell size, and the surrounding environment (Maller (1991)Curr. Opin. Cell Biol., 3:26). It is especially important formulti-cellular organisms to maintain integrity of genome, and there aremultiple checkpoints that monitor the state of genome. Among them are G1and G2 checkpoints prior to DNA replication and mitosis, respectively.It is crucial to repair or correct DNA damage before entering S phase,because once damaged DNA is replicated it often gives rise to mutations(Hartwell (1992) Cell, 71: 543). Progression through G1 and G2checkpoints without repairing extensive DNA damage induces mitoticcatastrophe and/or apoptosis.

Most cancer cells carry abnormalities in G1 checkpoint-related proteinssuch as p53, Rb, MDM-2, p16^(INK4) and p19^(ARF)(Levine (1997) Cell,88:323). Alternatively, mutations can cause overexpression and/orover-activation of oncogene products, e.g., Ras, MDM-2 and cyclin D,which reduce the stringency of G1 checkpoint. In addition to thesemutations, excessive growth factor signaling can be caused by theoverexpression of growth factors and can reduce the stringency of G1checkpoint. Together with loss-of-function and gain-of-functionmutations, continuous activation by growth factor receptors ordownstream signal-transducing molecules can cause cell transformation byoverriding the G1 checkpoint. A disrupted or abrogated G1 checkpointcontributes to higher mutation rates and the many mutations observed incancer cells. As a result, most cancer cells depend on G2 checkpoint forsurvival against excessive DNA damage (O'Connor and Fan (1996) Prog.Cell Cycle Res., 2:165).

The G2 cell cycle checkpoint restricts the onset of mitosis until DNAreplication and repair are complete. Malfunction of the G2 checkpointwould allow premature onset of mitosis prior to the completion of DNAreplication and repair, producing daughter cells lacking a substantialportion of the genomic DNA or harboring mutations. Functions of the G2checkpoint includes detecting DNA damage and generation of signal thatcan lead to cell cycle arrest when DNA damage is detected. The mechanismthat promotes the cell cycle G2 arrest after DNA damage is believed tobe conserved among species from yeast to human. In the presence ofdamaged DNA, Cdc2/Cyclin B kinase is kept inactive by phosphorylation ofthreonine-14 and tyrosine-15 residues on Cdc2 kinase; alternately, thelevel of Cyclin B protein may be reduced. At the onset of mitosis, Cdc25phosphatase removes inhibitory phosphates from Cdc2/Cyclin B kinase,thereby activating Cdc2/Cyclin B kinase. The activation of Cdc2/Cyclin Bkinase is equivalent to the onset of M phase.

In fission yeast, the protein kinase Chk1 is required for the cell cyclearrest in response to damaged DNA. Chk1 kinase acts downstream ofseveral rad gene products and is modified by the phosphorylation uponDNA damage. The kinases Rad53 of budding yeast and Cds1 of fission yeastare known to conduct signals from unreplicated DNA. It appears thatthere is some redundancy between Chk1 and Cds1 because elimination ofboth Chk1 and Cds1 culminated in disruption of the G2 arrest induced bydamaged DNA. Interestingly, both Chk1 and Cds1 phosphorylate Cdc25 andpromote Rad24 binding to Cdc25, which sequesters Cdc25 to cytosol andprevents Cdc2/Cyclin B activation. Therefore Cdc25 appears to be acommon target of these kinases implying that this molecule is anindispensable factor in the G2 checkpoint.

In humans, both hchk1, a human homologue of fission yeast Chk1, andChk2/HuCds1, a human homologue of the budding yeast Rad53 and fissionyeast Cds1, phosphorylate Cdc25C at serine-216, a critical regulatorysite, in response to DNA damage. This phosphorylation creates a bindingsite for small acidic proteins 14-3-3s, human homologues of Rad24 andRad25 of fission yeast. The regulatory role of this phosphorylation wasclearly indicated by the fact that substitution of serine-216 to alanineon Cdc25C disrupted cell cycle G2 arrest in human cells. However, themechanism of G2 checkpoint is not fully understood.

SUMMARY

This invention provides compounds that can be used to treat cellproliferation disorders, such as those associated with benign andmalignant cancer cells and further provides pharmaceutical compositionscontaining them. While the invention is not limited to any particularmechanism, it is believed that compounds of the invention can functionby inhibiting, disrupting, or abrogating the G2 checkpoint, inparticular by abrogating the DNA-damage-induced G2 checkpoint. Compoundsof the invention can act as anti-cancer agents by selectivelysensitizing cells with DNA damage, e.g., cancer cells, to the effects ofDNA damage. Compounds of the invention can sensitize cells, inparticular cancer cells, to the effects of DNA-damaging agents ortreatments. Compounds of the invention can suppress cell growth withoutany additional DNA-damaging treatment, and little or no cytotoxicactivity against normal cells. Thus, compounds of invention can be usedas anti-cancer agents, and as active ingredients in pharmaceuticalcompositions used as anti-cancer medicines, with or without anyadditional DNA-damaging treatment.

The invention provides methods for treating cells having proliferativedisorders. The invention provides a method for abrogating the G2checkpoint of a cell, in particular a method for abrogating theDNA-damage-induced G2 checkpoint, by contacting the cell with a compoundof the invention or a pharmaceutical composition of the invention in anamount sufficient to abrogate the G2 checkpoint. The invention furtherprovides a method for selectively sensitizing a cell with an impaired G1checkpoint to a DNA-damaging agent comprising, contacting the cell witha compound of the invention or a pharmaceutical composition of theinvention in an amount sufficient to abrogate the G2 checkpoint, therebysensitizing the cell to the DNA-damaging agent. The cell can be amammalian cell, in particular a human cell, more particularly a humancancer cell.

The invention provides a method for inducing mitotic catastrophe and/orapoptosis in a cell in an individual by administering a compound of theinvention or a pharmaceutical composition of the invention, in an amountsufficient to abrogate the G2 checkpoint in the cell and therebysensitizing the cell to a DNA-damaging agent, and administering aDNA-damaging agent. The cell can be a mammalian cell, in particular ahuman cell, in particular a human cancer cell. The cancer cell can havean impaired G1 cell cycle arrest checkpoint. The DNA-damaging agent canbe 5-fluorouracil (5-FU), rebeccamycin, adriamycin, bleomycin,cisplatin, hyperthermia, UV irradiation or gamma-irradiation, or anysuitable compounds that are known to cause DNA damage and/or areidentified by a screening method, e.g., as described in U.S. patentapplication Ser. No. 09/667,365.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of flow cytometry analysis of the DNA contentof Jurkat cells after the treatment with bleomycin (40 ug/ml), orbleomycin plus CBDC402 at various concentrations (0.2, 0.39, 0.78, 1.56,3.125, 6.25, 12.5, 25 and 50 ug/ml) for 24 hrs.

FIG. 2 shows the results of flow cytometry analysis of the DNA contentof the Jurkat cells after the treatment with colchicine (5 ug/ml), orcolchicine plus CBDC402 at various concentrations (0.2, 0.39, 0.78,1.56, 3.125, 6.25, 12.5, 25 and 50 ug/ml) for 24 hrs.

FIG. 3 a-b shows activities of various CBDC compounds; FIG. 3 a shows adose-response curve for G2 checkpoint abrogation by various CBDCcompounds and FIG. 3 b illustrates structure-activity relationships ofCBDC compounds.

FIG. 4 shows the structures, chemical names, and CBDC codes for certainCBDC compounds.

FIG. 5 shows the structure and relative activities of certain CBDCcompounds.

FIG. 6 shows the IC₅₀ values for G2 checkpoint abrogation by certainCBDC compounds.

FIG. 7 shows the results of flow cytometry analysis of the DNA contentof HCT116 cells after treatment with adriamycin (ADR), bleomycin (Bleo),comptothecin (Campto) and cisplatin (CDDP) for 24 hrs, after which timecells were treated with no CBDC004, or 2 uM, 10 uM, or 50 uM CBDC004.

FIG. 8 shows the results of flow cytometry analysis of cytoxicityresults (% subG1 population) for HCT116 cells treated with bleomycin (10ug/ml), adriamycin (1 ug/ml), camptothecine (1 ug/ml) or cisplatin(CDDP, 10 ug/ml), and within each treatment regime, cells were treatedwith no CBDC004, or 2 uM, 10 uM, or 50 uM CBDC004; the subG1 populationwas determined by staining cells with Krishan's solution.

FIG. 9 shows the effect on tumor growth of CPT-11, CBDC402, or acombination of CPT-11 and CBDC402, where human colon cancer cell lineHCT116 cells were subcutaneously implanted in SCID mice; mean tumorsizes for each treatment group were plotted (n=4) against the days aftertreatment.

FIG. 10 a-c shows results of flow cytometry analysis showing CBDC402specifically abrogates the DNA-damage-induced cell cycle G2 checkpoint;FIG. 10 a shows that CBDC402 abolishes the bleomycin-induced increase ofactivated normal T cells in G2 phase; FIG. 10 b shows that CBDC402abolishes the large bleomycin-induced increase of leukemic T cells(Jurkat cells) in G2 phase; FIG. 10 c shows that CBDC402 does not affectthe colchicine-induced increase of activated normal T cells in M phase.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for treating cellproliferation disorders. Specifically, the invention provides compoundsthat abrogate the cell cycle G2 checkpoint, which can be used to treatcell proliferation disorders such as those associated with cancer. Theinvention provides pharmaceutical compositions that contain one or morecompounds of the invention in a suitable carrier or excipient, whereinthese compositions may include additional active ingredients such asDNA-damaging agents. The invention provides methods for using compoundsof the invention, and pharmaceutical compositions containing compoundsof the invention, to suppress or kill proliferating cells, in particularcells with proliferation disorders. The invention further providesmethods for using compounds of the invention to selectively sensitize acell to the effects of other agents or treatments including DNA-damagingagents.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The term “abrogate the cell cycle G2 checkpoint” or “inhibit the cellcycle G2 checkpoint” or “disrupt the cell cycle G2 checkpoint” or“abrogation of the G2 checkpoint” any grammatical equivalent of theterm, refers to the ability of compounds of the invention to abrogatethe ability of a cell to arrest the cell cycle at the G2 checkpoint.Abrogation of cell cycle G2 checkpoint includes abrogation underconditions in the cell that otherwise would cause G2 cell cycle arrest,such as the accumulation of DNA damage by, e.g., certain anti-tumoragents, X-ray irradiation, gamma-ray irradiation, UV irradiation, orhyperthermia. Abrogation of the G2 checkpoint under such conditions isconsidered “abrogation of the G2 checkpoint” but more particularly,abrogation of “the DNA-damage-induced G2 checkpoint,” where it isunderstood that the DNA-damage-induced G2 checkpoint includesrecognition of DNA damage and generation of a signal that normallyproduces G2 cell cycle-arrest. A cell in which the cell cycle G2checkpoint is abrogated exhibits a decrease in the length of time thatthe cell is in the G2 checkpoint, which can range from absence of G2checkpoint altogether (G2 checkpoint arrest) to a G2 checkpoint having adecrease in duration of minutes, hours, days, weeks or longer underappropriate conditions. Thus, a cell contacted with a compound of theinvention has a G2 checkpoint time shorter in length than the cellnormally would have in the absence of the compound. For example, adecrease in the length of G2 checkpoint time would mean that a cellwhich is in G2 for a certain time, e.g., 4 hours, when contacted with aninvention compound, is in G2 for less than 4 hours, e.g., 3.5, 3, 2.5,2, 1 or fewer hours. The term “G2 abrogation” or “G2 checkpointabrogation” or “G2 checkpoint inhibitory activity” or any grammaticalequivalent, means any amount of abrogation or inhibition of the G2checkpoint.

As used herein, the term “apoptosis” refers to programmed cell death,and associated changes in cell physiology, including nucleic acidfragmentation, caspase activation, chromosome condensation, etc., as isunderstood in the art. The term “mitotic catastrophe” means cell deathresulting from an error in the mitotic process.

As used herein, the terms “DNA-damaging treatment” and “DNA-damagingagent” mean any treatment regimen that directly or indirectly damagesDNA. Specific examples of DNA-damaging agents include alkylating agents,nitrosoureas, anti-metabolites, plant alkaloids, plant extracts andradioisotopes. Specific examples of agents also include DNA-damagingdrugs, for example, 5-fluorouracil (5-FU), capecitabine, S-1 (Tegafur,5-chloro-2,4-dihydroxypyridine and oxonic acid), 5-ethynyluracil,arabinosyl cytosine (ara-C), 5-azacytidine (5-AC),2′,2′-difluoro-2′-deoxycytidine (dFdC), purine antimetabolites(mercaptopurine, azathiopurine, thioguanine), gemcitabine hydrochlorine(Gemzar), pentostatin, allopurinol, 2-fluoro-arabinosyl-adenine(2F-ara-A), hydroxyurea, sulfur mustard (bischloroetyhylsulfide),mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide,thiotepa, AZQ, mitomycin C, dianhydrogalactitol, dibromoducitol, alkylsulfonate (busulfan), nitrosoureas (BCNU, CCNU, 4-methyl CCNU or ACNU),procarbazine, decarbazine, rebeccamycin, anthracyclins such asdoxorubicin (adriamycin; ADR), daunorubibcin (Cerubicine), idarubicin(Idamycin) and epirubicin (Ellence), anthracyclin analogues such asmitoxantrone, actinimycin D, non intercalating topoisomerase inhibitorssuch as epipodophyllotoxins (etoposide=VP16, teniposide=VM-26),podophylotoxin, bleomycin (Bleo), pepleomycin, compounds that formadducts with nucleic acid including platinum derivatives, e.g.,cisplatin (CDDP), trans analogue of cisplatin, carboplatin, iproplatin,tetraplatin and oxaliplatin, as well as camptothecin, topotecan,irinotecan (CPT-11), and SN-38. Specific examples of nucleic aciddamaging treatments include radiation e.g., ultraviolet (UV), infrared(IR), or α-, β-, or γ-radiation, as well as environmental shock, e.g.,hyperthermia. One of skill in the art can identify and use otherDNA-damaging agents and treatments.

The term “compound of the invention” is intended to mean a molecule thathas the structure and activity disclosed herein. A compound of theinvention can be isolated, pure, substantially pure, or may be in acomposition containing a mixture of other components. Purity of acomposition containing a compound of the invention can be determined,for example, using analytical chemistry techniques such as highperformance liquid chromatography (HPLC). A composition as providedherein may contain one or more compounds of the invention, in a mixturewith suitable carriers, excipients, additional active ingredientsincluding DNA-damaging agents, and the like.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use, e.g., as a anti-cancer agent, in a subject. Thesubject may be a human in need of treatment for a cell proliferationdisorder. A pharmaceutical composition of the invention is a formulationthat comprises a pharmacologically effective amount of at least onecompound of the invention and a pharmaceutically acceptable carrier.

As used herein, the terms “proliferative disorder” and “proliferativecondition” mean any pathological or non-pathological physiologicalcondition characterized by aberrant or undesirable proliferation of atleast one cell, including conditions characterized by undesirable orunwanted cell proliferation or cell survival conditions characterized bydeficient or aberrant or deficient apoptosis, as well as conditionscharacterized by aberrant or undesirable or unwanted cell survival. Theterm “differentiative disorder” means any pathological ornon-pathological physiological condition characterized by aberrant ordeficient differentiation.

The term “subject” refers to animals, typically mammalian animals, suchas primates (humans, apes, gibbons, chimpanzees, orangutans, macaques),domestic animals (dogs and cats), farm animals (horses, cattle, goats,sheep, pigs) and experimental animals (mouse, rat, rabbit, guinea pig).Subjects include animal disease models (e.g., tumor bearing mice).

As used herein, the singular forms “a”, “and,” “the” and “is” includeplural referents unless the context clearly indicates otherwise. Thus,for example, reference to a “compound” includes a plurality of compoundsand reference to “a residue” or an “amino acid” includes reference toone or more residues and amino acids.

All publications, patents, and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

G2 Checkpoint Abrogation

While the invention is not limited to a particular mechanism of action,it has been observed that compounds of the invention can abrogate the G2checkpoint in proliferating cells. The G2 cell cycle checkpointrestricts the onset of mitosis until DNA replication and repair arecomplete, and disruption of the G2 checkpoint would allow prematureonset of mitosis prior to the completion of DNA replication and repair.Without wishing to be limited to this theory, it is believed that, incells that have accumulated DNA damage, abrogation of the G2 checkpointby compounds of the invention means the cells do not have an opportunityto correct or repair DNA damage at the G2 checkpoint and instead,proceed through G2 without DNA repair, which leads to mitoticcatastrophe, apoptosis, or other conditions resulting in cellsuppression or cell death.

In accordance with one aspect, the invention provides methods forabrogating the G2 cell cycle arrest induced by DNA damage. There aresignificant differences cell cycle responses, in particular in the G2checkpoint, in normal cells and in DNA-damaged cells. The damage-inducedG2 checkpoint includes recognition of DNA damage and generation of asignal that produces cell cycle-arrest. The invention provides compoundsthat selectively abrogate the DNA-damage-induced G2 checkpoint.

In accordance with another aspect, the invention provides compounds andpharmaceutical compositions that sensitize cells to DNA-damaging agentsand treatments. The invention provides methods for sensitizing cells toDNA-damaging agents and treatments.

In accordance with another aspect, the invention provides compounds andpharmaceutical compositions that selectively target cells with DNAdamage. The invention further provides methods for selectively targetingcells with DNA damage by contacting the cells with at least one compoundof the invention in an amount sufficient to abrogate DNA-damage-inducedG2 checkpoint. In one embodiment, cells with pre-existing DNA damage aretreated with compounds of the invention that abrogate the G2 checkpoint,and the DNA-damaged cells proceed through G2 phase, which results incell death or suppression (usually, mitotic catastrophe or apoptosis).In another embodiment, cells are treated a combination of at least oneDNA-damaging agent and at least one compound of the invention, resultingin a higher rate of cell death or suppression than the rate seen usingDNA-damaging agents alone.

In accordance with another aspect, the present invention providescompounds and pharmaceutical compositions that selectively target cellswith an impaired G1 cell cycle checkpoint, in particular cancer cells.The invention provides methods for selectively targeting cells with animpaired G1 cell cycle checkpoint, in particular cancer cells, bycontacting the cells with at least one compound of the invention in anamount sufficient to abrogate G2 cell cycle checkpoint. Without wishingto be limited by this theory, cells with an impaired G1 cell cyclecheckpoint do not repair DNA damage prior to G1, and abrogation of theG2 checkpoint by compounds of the present invention means these cellsproceed through mitosis without repairing accumulated DNA damage. Thelack of an effective G2 checkpoint after DNA damage becomes fatal to thecell having G1 checkpoint defect. If a cell progresses through G2without sufficient repair of DNA damage, the damage can lead to mitoticcatastrophe or apoptosis.

In accordance with one aspect, the present invention provides compoundsthat selectively target cancer cells, and kill or suppress growth ofcancer cells. The invention further provides methods for selectivelytargeting cancer cells, and killing or suppressing growth of cancercells, by contacting the cells with at least one compound of theinvention in an amount sufficient to abrogate the G2 checkpoint. Manycancer cells have mutations in genes involved in the G1 cell cyclearrest checkpoint, including impaired tumor suppressor genes such asp53, Rb, p16INK4, and p19ARF, and/or mutations that cause expression ofoncogenes such as MDM-2 and cyclin D. In addition, overriding the G1checkpoint can lead to transformation of normal cells into cancer cells,as excessive growth factor signaling caused by the overexpression ofgrowth factors, can lead to a condition wherein growth factor receptorsor downstream signal-transducing molecules cause cell transformation byoverriding the G1 checkpoint. In contrast, few cancers have disrupted G2cell cycle arrest checkpoints. Thus, the G2 checkpoint is usuallyretained in cancer cells with an impaired G1 checkpoint. Selectivedisruption of the G2 checkpoint would make cancer cells with an impairedG1 checkpoint more sensitive to DNA-damaging treatment, as compared tonormal cells with an intact G1 checkpoint, since progression through G1and G2 without repairing such damage induces apoptosis or mitoticcatastrophe. Without wishing to be limited to this theory, compounds ofthe present invention selectively disrupt (abrogate) the G2 checkpointin cancer cells, thereby causing cancer cells with an impaired G1checkpoint to be more sensitive to DNA-damaging treatment. Accordingly,the invention provides compounds that sensitize G1-checkpoint-impairedcancer cells to DNA-damaging agents and treatments.

In accordance with another aspect of the invention, compounds of theinvention can selectively target cancer cells with little or nocytotoxic effect on normal cells. Without wishing to be limited by thistheory, it is proposed that a normal cell in which the G2 checkpoint isabrogated by a compound of the invention will suffer little or nodeleterious consequences from entering G2 phase and undergoing mitosiswithout a functioning G2 checkpoint; in contrast, abrogation of theDNA-damaged G2 checkpoint in a DNA-damaged cell by a compound of theinvention is expected to have severe cytotoxic effects, leading toapoptosis or mitotic catastrophe. Thus, the invention provides methodsfor selectively targeting DNA-damaged cells such as cancer cells, withlittle or no cytotoxic effect on normal (undamaged) cells, by contactingthe cells with at least one compound of the invention in an amountsufficient to abrogate the G2 checkpoint. The provides pharmaceuticalcompositions containing at least one compound of the invention, suitablefor use in methods for selectively targeting targeting DNA-damaged cellssuch as cancer cells, with little or no cytotoxic effect on normal(undamaged) cells.

In accordance with yet another aspect of the invention, compounds of theinvention can selectively sensitize cells, in particular cancer cells,to the cell killing effects of DNA-damaging agents with little or nocytotoxic effect on normal cells. Most conventional anti-cancer agentstarget proliferating cells irrespective of whether they are cancer cellsor normal cells, with the result that most conventional anti-cancermedicines give rise to side effects such as nausea, diarrhea, or hairloss. In contrast, compounds of the present invention selectively targetcells with impaired G1 checkpoint, or other types of DNA damage, andtherefore have little or no cytotoxic effect on normal cells.

The invention provides a method for inducing apoptosis or mitoticcatastrophe in a cell by contacting the cell with at least one compoundof the invention in an amount sufficient to abrogate the G2 checkpoint.The invention further provides a method for inducing apoptosis ormitotic catastrophe in a cell by contacting the cell with at least onepharmaceutical composition of the invention in an amount sufficient toabrogate the G2 checkpoint. The cell can be a cell with DNA damage, inparticular a cancer cell.

The invention provides a method for inducing apoptosis or mitoticcatastrophe in a cell by contacting the cell with a DNA-damaging agentor treatment and at least one compound of the invention in an amountsufficient to abrogate the G2 checkpoint and thereby sensitize the cellto the DNA-damaging agent or treatment. The cell can be a cancer cell.The cancer cell can have an impaired G1 cell cycle checkpoint. TheDNA-damaging agent or treatment can be 5-flourouracil (5-FU),rebeccamycin, adriamycin, bleomcin, cisplatin, hyperthermia, UVirradiation, gamma-irradiation, or other DNA-damaging agent or treatmentsufficient to cause damage.

In accordance with one aspect, the invention provides a method forscreening for compounds capable of abrogating the G2 checkpoint by: (a)providing a test compound; (b) providing a population of cells; (c)administering to the population of cells an agent that causesaccumulation of G2/M phase cells after treatment; (d) administering thetest compound to a portion of the population treated with the agent thatcauses accumulation of G2/M phase cells; (e) measuring the number ofcells in G2/M phase in the population treated with the test compound;(f) measuring the number of cells in G2/M phase in the populationtreated only with the agent that causes accumulation of G2/M phasecells; (g) comparing the number in (e) with the number in (f) todetermine whether the test compound abrogated the G2 cell cyclecheckpoint. According to this method, accumulation of cells in G2/Mphase is used as an indicator of G2 cell cycle arrest. In oneembodiment, the agent induces DNA damage, and the method is useful forscreening for compounds capable of abrogating the DNA-damage-induced G2checkpoint. In another embodiment, the amount of DNA is measured usingflow cytometry, e.g., using propidium iodine to stain DNA and FACS™analysis, or the equivalent, to determine cell cycle stage by measuringthe DNA content of each cell. In one embodiment, the amount of DNA ismeasured after about 10 to about 72 hours after the contacting step.

The invention provides compounds that, when administered to a cell,abrogate the G2 checkpoint, in particular the DNA-damage-induced G2checkpoint, and kill or suppress cells, with or without DNA-damagingtreatment, wherein the compounds have the following general structure:

where either or both benzenes can be substituted with pyrazine,pyrimidine piperazine, morpholine, cyclohexane, piperizine or pyridine;R1 is a halogen such as bromine (Br), chlorine (Cl), fluorine (F),Iodine (I), amino (NH₂), nitro (NO₂), hydroxy (OH)O-methyl (OCH₃) methyl(CH₃) or hydrogen (H), R2, R3, R4, R5 and/or R6 is bromine (Br),chlorine (Cl), fluorine (F), Iodine (I), amino (NH₂), nitro (NO₂),methyl (CH₃), O-Methyl (OCH₃), hydroxy (OH), CH(CH₃)₂, CHO, CHOCH₃,O(CH₂)nCH₃, OCO(C₆H₁₂)Cl, COOCH₃ or hydrogen; X1 is nitrogen (NH),oxygen (O) or sulfate (S); X2 is oxygen (O) or sulfate (S). Illustrativeembodiments are found in the drawings, especially in FIGS. 3, 4, 5 and6, but compounds of the invention are not limited to these embodiments.

The invention provides compounds that, when administered to a cell,abrogate the G2 checkpoint, in particular the DNA-damage-induced G2checkpoint, and kill or suppress cells with or without DNA-damagingtreatment, wherein the compounds have the following general structure:

where either or both benzenes can be substituted with pyrazine,pyrimidine. piperazine, morpholine, cyclohexane, piperizine or pyridine;R1 is bromine (Br), chlorine (Cl), fluorine (F), Iodine (I), amino(NH₂), nitro (NO₂), hydroxy (OH)O-methyl (OCH₃) methyl (CH₃) or hydrogen(H), R2 is bromine (Br), chlorine (Cl), fluorine (F), Iodine (I), amino(NH₂), nitro (NO₂), methyl (CH₃), O-Methyl (OCH₃), hydroxy (OH),CH(CH₃)₂, CHO, CHOCH₃, O(CH₂)nCH₃, OCO(C₆H₁₂)Cl, COOCH₃ or hydrogen; X1is nitrogen (NH), oxygen (O) or sulfate (S); X2 is oxygen (O) or sulfate(S). Illustrative embodiments are found in the drawings, especially inFIGS. 3, 4, 5 and 6.

The invention provides compounds that abrogate the G2 checkpoint and/orsuppress or kill cancer cells, with or without DNA-damaging treatment,wherein the compounds have the following general structure:

wherein substitution of different molecules at positions R1 to R6affects the G2-checkpoint-abrogating activity of the resultingcompounds. The following structure-activity relations have beendetermined

At R1, bromine (Br) provides higher activity than chlorine (Cl),fluorine (F) or methyl (CH₃).

At R2, methyl (CH₃) or O-Methyl (OCH₃) provide higher activity thanhydroxide (OH), bromine (Br), Cloride (Cl), CH(CH₃)₂, CHO, CHOCH₃,O(CH₂)nCH₃, OCO(C₆H₁₂)CI or COOCH₃ or H.

At R3, methyl (CH₃) provided higher activity than H or O-Methyl (OCH₃).

At R4, bromine (Br), fluorine (F), chlorine (Cl), or H can be used.

At R5, bromine (Br), fluorine (F), chlorine (Cl), or H can be used.

At R6, bromine (Br), fluorine (F), chlorine (Cl), or H can be used.

The foregoing list is merely illustrative and not exhaustive.Illustrative embodiments are found in FIGS. 3, 4, 5, and 6. One of skillin the art can make additional substitutions and activity determinationsaccording to the teachings of the present disclosure, to obtainadditional compounds of the invention. It is understood by one of skillin the art that, although certain substitutions have been observed toproduce structures with higher activity than other structures withrespect to DNA-damage-induced G2 checkpoint abrogation, the inventionprovides compounds with all substitutions and all levels of activity.For a particular embodiment, one of skill will consider multiple factorsin selecting a compound of the invention for use in that embodiment, inaddition to the activity of a compound against a particular target. Oneof skill in the art will consider activity of the compound,availability, stability, ease or efficiency of synthesis, suitabilityfor formulation in a pharmaceutical composition, drugability,interaction with other compounds in vivo, ex vivo, or in vitro, abilityto kill cells, ability to suppress growth of cells, effects on normalcells, and other activities.

The invention provides compounds that abrogate the G2 checkpoint, inparticular compounds that selectively abrogate the DNA-damage-inducedcell cycle G2 checkpoint. Compounds are provided that selectivelyabrogate the DNA-damage-induced cell cycle G2 checkpoint in G1checkpoint-defective cells such as cancer cells, and selectivelyabrogate the DNA-damage-induced G2 checkpoint in cells treated withDNA-damaging agents. The invention provides compounds that sensitizecancer cells to DNA-damaging treatments. Further provided are compoundsthat inhibit xenograft tumor growth, alone or in combination withanti-cancer agents. The invention provides compounds that suppresscolony formation in vitro in cancer cells, alone or in combination withanti-cancer agents. In particular, the invention provides compounds thatabrogate the G2 checkpoint and/or suppress or kill cancer cells, with orwithout DNA-damaging treatment, including but not limited to:

CDBC004: 4-Chloro-benzoic acid 4-methoxy-phenyl ester

CBDC401: 4-Chloro-benzoic acid p-tolyl ester

CBDC402: 4-Bromo-benzoic acid p-tolyl ester

CBDC403: 3,4,5-Trifluoro-benzoic acid p-toly 1 ester

CBDC404: 4-Fluoro-benzoic acid 4-bromo-pheny 1 ester; compound withethane

CBDC405: 3,4-Dichloro-benzoic acid p-tolyl ester; compound with ethane

CBDC406: 2,4-Dichloro-benzoic acid p-tolyl ester; compound with ethane

CBDC407: 4-Fluoro-benzoic acid p-tolyl ester; compound with ethane

CBDC408: 2,3,4,5,6-Pentafluoro-benzoic acid p-tolyl ester; compound withethane

CBDC409: 4-Chloro-benzoic acid 3,4-dimethyl-phenyl ester; compound withethane

CBDC410: 4-Chloro-benzoic acid 4-hydroxy-phenyl ester; compound withethane

CBDC411: 4-Fluoro-benzoic acid 4-hydroxy-phenyl ester; compound withethane

CBDC412: 4-Bromo-benzoic acid 4-fluoro-phenyl ester

CBDC413: 4-Bromo-benzoic acid 4-trifluoromethyl-phenyl ester

CBDC414: 4-Bromo-benzoic acid 4-hydroxy-phenyl ester

CBDC415: 4-Bromo-benzoic acid 4-trifluoromethoxy-phenyl ester

CBDC418: 4-Bromo-benzoic acid 6-methyl-pyridin-3-yl ester

CBDC440: 4-Bromo-thiobenzoic acid O-p-tolyl ester

CBDC441: 4-Bromo-dithiobenzoic acid p-tolyl ester

CBDC442: 4-Bromo-thiobenzoic acid S-p-tolyl ester

Structures of these compounds are provided herein in FIGS. 3, 4, 5, and6, and in the claims.

The invention provides compositions that selectively abrogate theDNA-damage-induced cell cycle checkpoint. In one embodiment, compoundCBDC402 selectively abrogated the DNA-damage-induced cell cycle G2checkpoint in G1-checkpoint-defective cancer cells, as described belowin Example 1. Treatment of Jurkat cells (human T-cell leukemia-derivedcell line) with bleomycin, a DNA-damaging agent used as an anti-canceragent, resulted in accumulation of cells in the G2/M phase, indicatingG2 cell cycle arrest due to bleomycin-induced DNA damage. CBDC402abolished the bleomycin-induced accumulation of cells in G2/M in adose-dependent manner (FIG. 1). Colchicine does not induce G2 cell cyclearrest, and in another embodiment, CBDC402 did not inhibitcolchicine-induced accumulation of cells in G2/M phase cells at anyCBDC402 concentration (FIG. 2). Thus, CBDC402 selectively abrogated theDNA-damage-induced cell cycle checkpoint.

The invention provides compositions that abrogate DNA-damage-induced G2checkpoint when administered to a cell. In various embodiments,compounds CBDC004, CBDC402, CBDC403, CBDC404, CBDC405, CBDC406, CBDC407,CBDC408, CBDC409, CBDC410 and CBDC411 abrogated the G2 cell cyclecheckpoint in bleomycin-treated Jurkat cells in a dose-dependent manner(FIG. 3 a). A dose-response curve for G2 checkpoint abrogation byvarious CBDC compounds showed that CBDC402 showed the highest activityin this embodiment, and all CBDC compounds tested were capable ofabrogating G2 cell cycle checkpoint at the highest concentration (50μg/ml). The structures corresponding to different levels of activity areshown in FIG. 3 b; the CBDC designation corresponding to the structuresdisclosed in FIG. 3 b can be found by reference to FIG. 4.

The method provides compositions with different G2-checkpoint-abrogatingactivities, and further provides methods for determining theseactivities. In additional embodiments, CBDC compounds were tested fortheir G2 checkpoint abrogating activity. CBDC compounds were ranked ashighly active; moderately active; active; weakly active; and inactive,as shown in FIG. 5. The IC₅₀ of G2 checkpoint abrogation in Jurkat cellswas determined by dose-response studies carried out as described aboveactivity and ranked according to their activity, as shown in FIG. 6. InFIGS. 5 and 6, CBDC compounds are fully disclosed by disclosure of theirchemical structures, and in some entries, the CBDC compounds areadditionally identified by a CBDC designation number.

The invention provides compositiions that abrogate theDNA-damage-induced G2 checkpoint induced by a variety of DNA-damagingagents. In one embodiment, compound CBDC004 abrogated theDNA-damage-induced G2 checkpoint activated by various anti-canceragents. Human cancer cells (HCT116 human colon carcinoma cells) weretreated with bleomycin, adriamycin, camptothecin, or cisplatin (CDDP),with or without compound CBDC004. Each of these anti-cancer agentsinduced an accumulation of cells at cell cycle G2/M, and co-incubationwith CBDC004 reduced the number of cells at G2/M, indicating thatCBDC004 had abrogated the DNA-damage-induced G2 checkpoint activated bybleomycin, adriamycin, camptothecin, or cisplatin.

The invention provides compositions and methods for sensitizing cells toDNA-damaging treatments. In yet another embodiment, CBDC004 sensitizedhuman cells to the cytotoxic effects of various DNA-damaging treatmentsthat are used as anti-cancer agents. Human cancer cells (HCT116 cells)were treated with bleomycin, adriamycin, canptothecine or cisplatin,with or without CBDC004, and the number of dead cells was measured afterthe treatment. CBDC004 sensitized the cells to the cytotoxic effects ofeach DNA-damaging treatment (FIG. 8). Administering compounds of thepresent invention to cells sensitizes the cells to DNA-damagingtreatments, making the DNA-damaging treatments more effective.

The invention provides compositions and methods for inhibiting growth ofxenograft tumors. In another embodiment, CBDC402 inhibited growth ofxenograft tumors. After HCT-116 human colon carcinoma cells wereimplanted subcutaneously in Severe Combined Immunodeficiency (SCID)mice, various compounds were administered and tumor growth wasmonitored. CBDC402 alone gave a slight reduction in tumor growth, andcombinations of CBDC402 plus CPT-11 (CAMPTOSAR®, Irinotecan, atopoisomerase inhibitor) significantly reduced or inhibited tumorgrowth.

The invention provides compositions and methods for treating cells withproliferative disorders. In particular, the invention provides compoundsand methods for inhibiting various aspects of cells having proliferativedisorders, including inhibiting colony formation by cancer cells invitro. Compounds of the invention inhibited colony formation by cancercells in vitro, alone or in combination with an anti-cancer agent. Inone embodiment, MK-45 cells from a human gastric cancer derived cellline, seeded into multiwell plates, were treated with CBDC402, CBDC412,CBDC413, and CBDC418. Treatment with CBDC402 alone caused significantsuppression of colony formation by MK-45 cells, and treatment withCBDC412 caused a slight suppression of colony formation. In anembodiment that also included CPT-11, addition of CBDC402 appeared toaugment the effect of CPT-11, resulting in almost complete suppressionof colony formation.

The invention provides compositions and methods to selectively abrogatethe DNA-damage-induced G2 checkpoint without affecting the M checkpoint.In one embodiment, CBDC402 selectively abrogates the DNA-damage-inducedG2 checkpoint. Bleomycin induced a moderate increase in the accumulationof activated normal T cells in G2 phase (FIG. 10 a), and induced a largeaccumulation of cells in Jurkat cells (leukemic T cells) at G2 phase(FIG. 10 b). CBDC abolished the bleomycin-induced increase of cells inG2 phase in both cell lines (FIG. 10 a,b). When activated normal T cellsreceived were treated with colchicine, which caused an accumulation ofcells in M phase, and CBDC402 did not affect the colchicine-inducedincrease of activated normal T cells at M phase. This embodimentdemonstrates that CBDC402 selectively abrogates the G2 cell cyclecheckoint and not the not M phase checkpoint.

The invention provides methods for synthesizing compounds of theinvention. In one embodiment, CBDC 412 (4-bromo-benzoic acid4-fluoro-phenyl ester) is synthesized as described below. In oneembodiment, ten ml of dioxane, 5 mmol (1.1 g) of 4-bromo-benzoic acidchlorine, and 5 mmol (0.56 g) of 4-fluoro-phenol were added to 50 mlfour-mouth-flask sequentially and dissolved at room temperature.Triethylamine dissolved in dioxane was slowly dripped into this solutionand the solution was stirred for three hours at room temperature. Theprecipitated crystals were filtered and extracted with benzene. Theextracted solution was washed with sodium hydrocarbonate several times,magnesium anhydrate was added, and the resulting solution was dried andfiltered. The solution was distilled under low pressure andcrystallized. The row crystals were yellowish-white, weighing 1.37 g. Aportion of this crystal (0.5 g) was dissolved in benzene and purifiedwith 10 g of silica gel. The purified product was white, and the puritywas confirmed to 99.93% by liquid chromatography (LC). The structure wasconfirmed by NMR (see, Example 6).

Screening

The invention provides compositions and methods for screening forpotential therapeutic compounds (“test compounds” or “candidatecompounds”) to inhibit or abrogate the G2 checkpoint. Assay formats thatcan be used for screening are well known. For a general description ofdifferent formats for binding assays, see, e.g., BASIC AND CLINICALIMMUNOLOGY, 7th Ed., Stiles and Terr, eds.(1991); ENZYME IMMUNOASSAY,Maggio, ed., CRC Press, Boca Raton, Fla. (1980); and “Practice andTheory of Enzyme Immunoassays” in Tijssen, LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY, Elsevier Science Publishers, B.V.Amsterdam (1985).

Targets

Subjects appropriate for treatment include those currently undergoing orare candidates for treatment for a proliferative or differentiativedisorder or (e.g., anti-tumor therapy). Additional candidate subjectsinclude, for example, subjects at risk of developing a cellproliferative disorder. The invention methods are therefore applicableto treating a subject who is at risk of developing a cell proliferativedisorder but who has not yet exhibited overt symptoms of the disorder.At risk subjects can be identified as having a genetic predisposition orfamily history to developing a cell proliferative disorder. For example,subjects having an activated oncogene or having a mutation or deletionof a tumor suppressor gene are candidate subjects. At risk subjects cantherefore be identified using routine genetic screening for the presenceof the genetic lesion, or inquiry into the subjects' family history toestablish that they are at risk of the disorder. A particular example ofan at risk subject would be one with a family history or other geneticcharacteristic indicating predisposition to a cancer in which theneoplastic or drug-resistant neoplastic cells express CD40. A particularspecific example of a genetic disease is retinoblastoma, which is causedby a defect in the Rb tumor suppressor gene.

Typically an “effective amount” or “sufficient amount” of a compound ofthe invention is administered, where that is an amount sufficient toproduce the desired affect. Effective amounts therefore are determinedby measuring one or more of: decreasing cell proliferation, decreasingnumbers of cells, inhibiting increased proliferation, inhibitingincreased numbers of cells, increasing apoptosis, or decreasingsurvival, of at least a portion of the cells comprising theproliferating cells (e.g., at least some of the target cells). Thus, forexample, where it is desired to inhibit cell proliferation, an effectiveamount will be an amount that detectably decreases cell proliferation ornumbers of proliferating cells, or increases cell apoptosis or decreasescell survival. The amount can therefore be sufficient to reduce targetcell numbers, stabilize target cell numbers or inhibit increases intarget cell numbers. For example, where the disorder comprises a solidtumor, reducing tumor size, stabilizing tumor size, or preventingfurther growth of the tumor, of at least a portion of the tumor (e.g.inhibiting growth of 5-10% of the cells, or 10-20% or more of the cellscomprising the tumor mass) is a satisfactory clinical endpoint. Wherethe disorder comprises a liquid tumor, reducing numbers of tumor cells,stabilizing tumor cell numbers or inhibiting further increases in tumorcell numbers, of at least a subpopulation of the tumor cells (e.g.inhibiting growth of 5-10% of the cells, or 10-20% or more of the cells)is a satisfactory clinical endpoint.

In addition, amounts considered effective can prevent or inhibitprogression of the condition or disorder. For example, certain tumors asthey progress become increasingly aggressive, including progressing tometastatic forms. Thus, amounts also considered effective would resultin reducing or preventing the tumors from becoming increasinglyaggressive or from metastasizing. Accordingly, inhibiting or preventinga worsening of the disorder or condition, i.e., stabilizing thecondition is an additional satisfactory clinical endpoint.

Examination of a biological sample containing a liquid tumor (e.g.,blood or a tissue sample), can establish whether tumor cell mass ornumbers have been reduced, or inhibition of tumor cell proliferation hasoccurred. For a solid tumor, invasive and non-invasive imaging methodscan ascertain a reduction in tumor size, or inhibiting increases in thetumor size. Decreasing counts of receptor of a receptor positive tumor,can be used to assess reduction or inhibition of tumor cellproliferation. Amounts of hormone of a hormone producing tumor, e.g.,breast, testicular, or ovarian cancers, can be used to assess areduction or inhibition of proliferation of the tumor.

Effective amounts can also objectively or subjectively reduce ordecrease the severity or frequency of symptoms associated with thedisorder or condition. For example, an amount of an invention compoundthat reduces pain, nausea or other discomfort, or increases appetite orsubjective well being is a satisfactory clinical endpoint.

Effective amounts also include a reduction of the amount (e.g., dosage)or frequency of treatment with another protocol, which is considered asatisfactory clinical endpoint. For example, a cancer patient treatedwith an invention compound may require less nucleic acid damagingtreatment in order to inhibit cancer cell proliferation. In thisexample, an effective amount would include an amount that reduces thedosage frequency or amount of a nucleic acid damaging agent that thesubject is administered in comparison to the dosage frequency or amountadministered without treatment with a compound of the invention.

Methods of the invention that lead to an improvement in the subject'scondition or a therapeutic benefit may be relatively short in duration,e.g., the improvement may last several hours, days or weeks, or extendover a longer period of time, e.g., months or years. An effective amountneed not be a complete ablation of any or all symptoms of the conditionor disorder. Thus, a satisfactory clinical endpoint for an effectiveamount is achieved when there is a subjective or objective improvementin the subjects' condition as determined using any of the foregoingcriteria or other criteria known in the art appropriate for determiningthe status of the disorder or condition, over a short or long period oftime. An amount effective to provide one or more beneficial effects, asdescribed herein or known in the art, is referred to as an “improvement”of the subject's condition or “therapeutic benefit” to the subject.

An effective amount of an invention compound can be determined basedupon animal studies or optionally in human clinical trials. The skilledartisan will appreciate the various factors that may influence thedosage and timing required to treat a particular subject including, forexample, the general health, age, or gender of the subject, the severityor stage of the disorder or condition, previous treatments,susceptibility to undesirable side effects, clinical outcome desired andthe presence of other disorders or conditions. Such factors mayinfluence the dosage and timing required to provide an amount sufficientfor therapeutic benefit. The dosage regimen also takes intoconsideration the pharmacokinetics, i.e., the pharmaceuticalcomposition's rate of absorption, bioavailability, metabolism, andclearance. In addition, doses or treatment protocols may be specificallytailored to the subject or modified based on pharmacogenomic data.

Cells that may be treated with the compounds of the invention includeany cell whose proliferation it is desired to inhibit or prevent invitro, ex vivo or in vivo. Certain target cells exhibit a shorter thannormal cell cycle G1 checkpoint time or have an impaired cell cycle G1checkpoint such that the cells exit the G1 checkpoint before enough timehas passed to complete nucleic acid repair. Candidate cells can also beidentified by contacting a test cell with an invention compound alone,or in combination with a DNA-damaging treatment, and determining if thecontacted cell exhibits decreased proliferation or increased cell death,in particular apoptosis or mitotic catastrophe.

Invention compounds are therefore useful for inhibiting cellproliferation in vitro, ex vivo and in vivo. As such, subjects having orat risk of having a disorder or physiological condition characterized byabnormal or undesirable or unwanted cell proliferation or cell survival,or abnormal or deficient cell differentiation, can be treated with acompound of the invention alone or in combination with a treatment thatdirectly or indirectly causes DNA damage, or in combination with ananti-proliferative treatment.

Thus, in accordance with the invention, there are provided methods forinhibiting cell proliferation, methods for increasing sensitivity of acell to a DNA-damaging agent or treatment and methods for increasingnucleic acid damage to a cell in vitro, ex vivo and in vivo. In oneembodiment, a method includes contacting a cell (e.g., a cultured cellor a cell present in a subject) with an amount of compound of theinvention sufficient to abrogate the G2 checkpoint. In anotherembodiment, a method includes contacting the cell with an amount of acompound of the invention sufficient to increase sensitivity of the cellto a DNA-damaging agent or treatment. In yet another embodiment, amethod includes contacting a cell with an amount of a compound of theinvention sufficient to increase nucleic acid damage of the cell. Invarious aspects, a method further includes contacting the cell with aDNA-damaging agent or exposing the cell to a DNA-damaging treatment.

Further provided are methods of treating a cell proliferative disorderor differentiative disorder in a subject, including conditionscharacterized by undesirable or unwanted cell proliferation or cellsurvival, conditions characterized by deficient or aberrant apoptosis,conditions characterized by aberrant or deficient cell survival, as wellas conditions characterized by aberrant or deficient celldifferentiation. In one embodiment, a method includes administering to asubject having or at risk of having a cell proliferative disorder, anamount of a compound of the invention effective to treat the cellproliferative disorder. In one aspect, the amount is sufficient toimprove the subject's condition. In particular aspects, the improvementincludes, in at least a portion of the target cells (e.g., abnormallyproliferating cells), decreased cell proliferation, decreased numbers ofcells, inhibiting increases in the number of cells, increased apoptosis,or decreased survival. In yet another aspect, a compound of theinvention is administered to a subject prior to, contemporaneously with,or after administering a treatment that inhibits cell proliferation. Inadditional particular aspects, at least a part of the cells of the cellproliferative disorder are located in blood, breast, lung, thyroid, heador neck, brain, lymph, gastrointestinal tract, genito-urinary tract,kidney, pancreas, liver, bone, muscle, or skin.

In another embodiment, a method includes administering an amount ofcompound of the invention to a subject to treat a solid tumor. In yetanother embodiment, a method includes administering an amount ofcompound of the invention to the subject to treat a liquid tumor. Invarious aspects, the subject having the tumor is administered with ancompound of the invention prior to, contemporaneously with, or afteranother anti-tumor therapy.

Use of Compounds of the Invention to Treat Proliferative orDifferentiative Disorders.

Proliferative or differentiative disorders amenable to treatment usingcompositions and methods provided herein include diseases andnon-pathological physiological conditions, both benign and neoplastic,characterized by abnormal or undesirable cell numbers, cell growth orcell survival. Such disorders or conditions may therefore constitute adisease state and include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, or may be non-pathologic, i.e., a deviation from normal butwhich is not typically associated with disease. A specific example of anon-pathologic condition that may be treated in accordance with theinvention is tissue re-growth from wound repair that results inscarring.

Cells comprising the proliferative or differentiative disorder may beaggregated in a cell mass or be dispersed. The term “solid tumor” refersto neoplasias or metastases that typically aggregate together and form amass. Particular examples include visceral tumors such as gastric orcolon cancer, hepatomas, venal carcinomas, lung and braintumors/cancers. A “liquid tumor” refers to neoplasias of thehaematopoetic system, such as lymphomas, myelomas and leukemias, orneoplasias that are diffuse in nature, as they do not typically form asolid mass. Particular examples of leukemias include acute and chroniclymphoblastic, myeolblasitc and multiple myeloma.

Such disorders include neoplasms or cancers, which can affect virtuallyany cell or tissue type, e.g., carcinoma, sarcoma, melanoma, metastaticdisorders or haematopoietic neoplastic disorders. A metastatic tumor canarise from a multitude of primary tumor types, including but not limitedto breast, lung, thyroid, head and neck, brain, lymphoid,gastrointestinal (mouth, esophagus, stomach, small intestine, colon,rectum), genito-urinary tract (uterus, ovary, cervix, bladder, testicle,penis, prostate), kidney, pancreas, liver, bone, muscle, skin, etc.

Carcinomas refer to malignancies of epithelial or endocrine tissue, andinclude respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas,breast carcinomas, prostatic carcinomas, endocrine system carcinomas,and melanomas. Exemplary carcinomas include those forming from thecervix, lung, prostate, breast, head and neck, colon, liver and ovary.The term also includes carcinosarcomas, e.g., which include malignanttumors composed of carcinomatous and sarcomatous tissues. Adenocarcinomaincludes a carcinoma of a glandular tissue, or in which the tumor formsa gland like structure.

Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplarysarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma,and fibrosarcoma.

As used herein, the term “haematopoietic proliferative disorder” means adisease involving hyperplastic/neoplastic cells of haematopoieticorigin, e.g., arising from myeloid, lymphoid or erythroid lineages, orprecursor cells thereof. Typically, the diseases arise from poorlydifferentiated acute leukemias, e.g., erythroblastic leukemia and acutemegakaryoblastic leukemia. Additional exemplary myeloid disordersinclude, but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CML);lymphoid malignancies include, but are not limited to, acutelymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional malignant lymphomas include, but are not limited to,non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Treatments for use in combination with compounds of the inventioninclude any anti-proliferative, DNA-damaging, or anti-tumor treatment asdisclosed herein or known in the art. For example, an anti-cellproliferative or anti-tumor treatment may comprise radiation treatmentor surgical resection optionally in combination with drug treatment. Thetreatment may comprise administration of a chemical substance, such as aradioisotope, a drug, such as a chemotherapeutic agent, or genetictherapy, such as an anti-oncogene (e.g., Rb, DCC, p53, etc.), a dominantnegative oncogene or an antisense to an oncogene. The compounds can beadministered prior to, contemporaneously with or following othertreatment protocols. For example, a candidate subject for anti-cellproliferative therapy (e.g., radiation therapy, chemotherapy, genetherapy, surgical resection, etc.) can be administered an inventioncompound prior to initiating the anti-cell proliferative therapy. Thus,prophylactic treatment methods are provided.

Combinatorial Chemical Libraries

Combinatorial chemical libraries are one means to assist in thegeneration of new chemical compound leads, i.e., compounds that inhibitor abrogate the G2 cell cycle arrest checkpoint. A combinatorialchemical library is a collection of diverse chemical compounds generatedby either chemical synthesis or biological synthesis by combining anumber of chemical “building blocks” such as reagents. For example, alinear combinatorial chemical library such as a polypeptide library isformed by combining a set of chemical building blocks called amino acidsin every possible way for a given compound length (i.e., the number ofamino acids in a polypeptide compound). Millions of chemical compoundscan be synthesized through such combinatorial mixing of chemicalbuilding blocks. For example, the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds. Preparation and screening of combinatorial chemical librariesare well known to those of skill in the art, see, e.g., U.S. Pat. Nos.6,004,617; 5,985,356. Such combinatorial chemical libraries include, butare not limited to, peptide libraries (see, e.g., U.S. Pat. No.5,010,175; Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghtonet al. (1991) Nature, 354: 84-88). Other chemistries for generatingchemical diversity libraries include, but are not limited to: peptoids(see, e.g., WO 91/19735), encoded peptides (see, e.g., WO 93/20242),random bio oligomers (see, e.g., WO 92/00091), benzodiazepines (see,e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins,benzodiazepines and dipeptides (see, e.g., Hobbs (1993) Proc. Nat. Acad.Sci. USA 90: 6909 6913), vinylogous polypeptides (see, e.g., Hagihara(1992) J. Amer. Chem. Soc. 114: 6568), non-peptidal peptidomimetics witha Beta D Glucose scaffolding (see, e.g., Hirschmann (1992) J. Amer.Chem. Soc. 114: 9217 9218), analogous organic syntheses of smallcompound libraries (see, e.g., Chen (1994) J. Amer. Chem. Soc. 116:2661), oligocarbamates (see, e.g., Cho (1993) Science 261:1303), and/orpeptidyl phosphonates (see, e.g., Campbell (1994) J. Org. Chem. 59:658). See also Gordon (1994) J. Med. Chem. 37:1385; for nucleic acidlibraries, peptide nucleic acid libraries, see, e.g., U.S. Pat. No.5,539,083; for antibody libraries, see, e.g., Vaughn (1996) NatureBiotechnology 14:309-314; for carbohydrate libraries, see, e.g., Lianget al. (1996) Science 274: 1520-1522, U.S. Pat. No. 5,593,853; for smallorganic molecule libraries, see, e.g., for isoprenoids U.S. Pat. No.5,569,588; for thiazolidinones and metathiazanones, U.S. Pat. No.5,549,974; for pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; formorpholino compounds, U.S. Pat. No. 5,506,337; for benzodiazepines U.S.Pat. No. 5,288,514.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., U.S. Pat. Nos. 6,045,755; 5,792,431; 357 MPS, 390MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn,Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus,Millipore, Bedford, Mass.). A number of robotic systems have also beendeveloped for solution phase chemistries. These systems includeautomated workstations, e.g., like the automated synthesis apparatusdeveloped by Takeda Chemical Industries, LTD. (Osaka, Japan) and manyrobotic systems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; Orca, Hewlett Packard, Palo Alto, Calif.) which mimicthe manual synthetic operations performed by a chemist. Any of the abovedevices are suitable for use with the present invention. The nature andimplementation of modifications to these devices (if any) so that theycan operate as discussed herein will be apparent to persons skilled inthe relevant art. In addition, numerous combinatorial libraries arethemselves commercially available (see, e.g., ComGenex, Princeton, N.J.,Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow,RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md.,etc.).

Formulation and Administration of Pharmaceutical Compositions

In one embodiment, the compounds of the invention are combined with apharmaceutically acceptable carrier (excipient) to form apharmacological composition. Pharmaceutically acceptable carriers cancontain a physiologically acceptable compound that acts to, e.g.,stabilize, or increase or decrease the absorption or clearance rates ofthe pharmaceutical compositions of the invention. Physiologicallyacceptable compounds can include, e.g., carbohydrates, such as glucose,sucrose, or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins,compositions that reduce the clearance or hydrolysis of the compounds,or excipients or other stabilizers and/or buffers. Detergents can alsoused to stabilize or to increase or decrease the absorption of thepharmaceutical composition, including liposomal carriers.Pharmaceutically acceptable carriers and formulations for compounds asdisclosed herein are known to the skilled artisan and are described indetail in the scientific and patent literature, see e.g., the latestedition of Remington's Pharmaceutical Science, Mack Publishing Company,Easton, Pa. (“Remington's”).

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives which areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, e.g.,phenol and ascorbic acid. One skilled in the art would appreciate thatthe choice of a pharmaceutically acceptable carrier including aphysiologically acceptable compound depends, for example, on the routeof administration of the compound of the invention and on its particularphysio-chemical characteristics.

In one embodiment, a solution of at least one compound of the inventionis dissolved in a pharmaceutically acceptable carrier, e.g., an aqueouscarrier if the composition is water-soluble. Examples of aqueoussolutions that can be used in formulations for enteral, parenteral ortransmucosal drug delivery include, e.g., water, saline, phosphatebuffered saline, Hank's solution, Ringer's solution, dextrose/saline,glucose solutions and the like. The formulations can containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents, detergents and the like. Additives canalso include additional active ingredients such as bactericidal agents,or stabilizers. For example, the solution can contain sodium acetate,sodium lactate, sodium chlorine, potassium chlorine, calcium chlorine,sorbitan monolaurate or triethanolamine oleate. These compositions canbe sterilized by conventional, well-known sterilization techniques, orcan be sterile filtered. The resulting aqueous solutions can be packagedfor use as is, or lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration. Theconcentration of peptide in these formulations can vary widely, and willbe selected primarily based on fluid volumes, viscosities, body weightand the like in accordance with the particular mode of administrationselected and the patient's needs.

Solid formulations can be used for enteral (oral) administration. Theycan be formulated as, e.g., pills, tablets, powders or capsules. Forsolid compositions, conventional nontoxic solid carriers can be usedwhich include, e.g., pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like. For oral administration, apharmaceutically acceptable nontoxic composition is formed byincorporating any of the normally employed excipients, such as thosecarriers previously listed, and generally 10% to 95% of activeingredient (e.g., peptide). A non-solid formulation can also be used forenteral administration. The carrier can be selected from various oilsincluding those of petroleum, animal, vegetable or synthetic origin,e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like.Suitable pharmaceutical excipients include e.g., starch, cellulose,talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, magnesium stearate, sodium stearate, glycerol monostearate,sodium chlorine, dried skim milk, glycerol, propylene glycol, water,ethanol.

Compounds of the invention, when administered orally, can be protectedfrom digestion. This can be accomplished either by complexing thecompound or compounds with a composition to render it resistant toacidic and enzymatic hydrolysis or by packaging the peptide or complexin an appropriately resistant carrier such as a liposome. Means ofprotecting compounds from digestion are well known in the art, see,e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm.Pharmacol. 48:119 135; U.S. Pat. No. 5,391,377, describing lipidcompositions for oral delivery of therapeutic agents (liposomal deliveryis discussed in further detail, infra).

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art, andinclude, e.g., for transmucosal administration, bile salts and fusidicacid derivatives. In addition, detergents can be used to facilitatepermeation. Transmucosal administration can be through nasal sprays orusing suppositories. See, e.g., Sayani (1996) “Systemic delivery ofpeptides and proteins across absorptive mucosae” Crit. Rev. Ther. DrugCarrier Syst. 13:85 184. For topical, transdermal administration, theagents are formulated into ointments, creams, salves, powders and gels.Transdermal delivery systems can also include, e.g., patches.

Compounds of the invention can also be administered in sustaineddelivery or sustained release mechanisms, which can deliver theformulation internally. For example, biodegradeable microspheres orcapsules or other biodegradeable polymer configurations capable ofsustained delivery of a compound as disclosed herein can be included inthe formulations of the invention (see, e.g., Putney (1998) Nat.Biotechnol. 16:153 157).

For inhalation, compounds of the invention can be delivered using anysystem known in the art, including dry powder aerosols, liquids deliverysystems, air jet nebulizers, propellant systems, and the like. Forexample, the pharmaceutical formulation can be administered in the formof an aerosol or mist. For aerosol administration, the formulation canbe supplied in finely divided form along with a surfactant andpropellant. In another embodiment, the device for delivering theformulation to respiratory tissue is an inhaler in which the formulationvaporizes. Other liquid delivery systems include, e.g., air jetnebulizers.

In preparing pharmaceuticals of the present invention, a variety offormulation modifications can be used and manipulated to alterpharmacokinetics and biodistribution. A number of methods for alteringpharmacokinetics and biodistribution are known to one of ordinary skillin the art. Examples of such methods include protection of the complexesin vesicles composed of substances such as proteins, lipids (forexample, liposomes, see below), carbohydrates, or synthetic polymers(discussed above). For a general discussion of pharmacokinetics, see,e.g., Remington's, Chapters 37-39.

The compounds and compositions used in the methods of the invention canbe delivered alone or as pharmaceutical compositions by any means knownin the art, e.g., systemically, regionally, or locally (e.g., directlyinto, or directed to, a tumor); by intraarterial, intrathecal (IT),intravenous (IV), parenteral, intra-pleural cavity, topical, oral, orlocal administration, as subcutaneous, intra-tracheal (e.g., by aerosol)or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasalmucosa). Actual methods for preparing administrable compositions will beknown or apparent to those skilled in the art and are described indetail in the scientific and patent literature, see e.g., Remington's.For a “regional effect,” e.g., to focus on a specific organ, one mode ofadministration includes intra-arterial or intrathecal (IT) injections,e.g., to focus on a specific organ, e.g., brain and CNS (see e.g., Gurun(1997) Anesth Analg. 85:317 323). For example, intra-carotid arteryinjection if preferred where it is desired to deliver a peptide orpolypeptide complex of the invention directly to the brain. Parenteraladministration is a preferred route of delivery if a high systemicdosage is needed. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in detail, in e.g., Remington's,. See also,Bai (1997) J. Neuroimmunol. 80:65 75; Warren (1997) J. Neurol. Sci.152:31 38; Tonegawa (1997) J. Exp. Med. 186:507 515.

In one embodiment, the pharmaceutical formulations comprising compoundsof the invention are incorporated in lipid monolayers or bilayers, e.g.,liposomes, see, e.g., U.S. Pat. Nos. 6,110,490; 6,096,716; 5,283,185;5,279,833. Liposomal formulations can be by any means, includingadministration intravenously, transdermally (see, e.g., Vutla (1996) J.Pharm. Sci. 85:5 8), transmucosally, or orally. The invention alsoprovides pharmaceutical preparations in which the peptides and/orcomplexes of the invention are incorporated within micelles and/orliposomes (see, e.g., Suntres (1994) J. Pharm. Pharmacol. 46:23 28;Woodle (1992) Pharm. Res. 9:260 265). Liposomes and liposomalformulations can be prepared according to standard methods and are alsowell known in the art, see, e.g., Remington's; Akimaru (1995) CytokinesMol. Ther. 1:197 210; Alving (1995) Immunol. Rev. 145:5 31; Szoka (1980)Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728and 4,837,028.

A pharmaceutically acceptable formulation can incorporate about 1% to99.9% of active ingredient (e.g., a compound of the present invention).The pharmaceutical compositions can be sterilized by conventional,well-known sterilization techniques, or can be sterile filtered.Additional pharmaceutical formulations and delivery systems are known inthe art and are applicable in the methods and compositions of theinvention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18thed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed.,Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles ofSolid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa.,(1993); and Poznansky et al., Drug Delivery Systems, R. L. Juliano, ed.,Oxford, N.Y. (1980), pp. 253-315)

The pharmaceutical formulations can be packaged in unit dosage form forease of administration and uniformity of dosage. “Unit dosage form” asused herein refers to physically discrete unitary dosages foradministration to the subject to be treated; each unit contains apredetermined quantity of compound that produces a desired effect incombination with a pharmaceutical carrier or excipient.

The invention further provides kits including invention compounds andpharmaceutical formulations thereof, optionally packaged into suitablepackaging material. A kit typically includes a label or packaging insertincluding a description of the components or instructions for use invitro, in vivo, or ex vivo, of the components therein. A kit can containa collection of such components, e.g., two or more invention compoundsor an invention compound in combination with a nucleic acid damagingagent or an anti-proliferative agent.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). The label or packaging insert can include appropriate writteninstructions. Kits of the invention therefore can additionally includelabels or instructions for using the kit components in any method of theinvention. Instructions can include instructions for practicing any ofthe methods of the invention described herein including treatment,detection, monitoring or diagnostic methods. Thus, for example, a kitcan include an invention compound in a pack, or dispenser together withinstructions for administering the compound in a treatment method of theinvention. Instructions may additionally include indications of asatisfactory clinical endpoint or any adverse symptoms that may occur,or additional information required by regulatory agencies such as theFood and Drug Administration for use on a human subject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin or affixed to the kit, or on a label affixed to the kit orpackaging material, or attached to a vial or tube containing a componentof the kit. Instructions may additionally be included on a computerreadable medium, such as a disk (floppy diskette or hard disk), opticalCD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storage mediasuch as RAM and ROM, IC tip and hybrids of these such asmagnetic/optical storage media.

Invention kits can additionally include a buffering agent, or apreservative or a stabilizing agent in a pharmaceutical formulation.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package.Invention kits can be designed for cold storage.

Treatment Regimens: Pharmacokinetics

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. Dosages fortypical pharmaceutical compositions are well known to those of skill inthe art. Such dosages are typically advisorial in nature and areadjusted depending on the particular therapeutic context, patienttolerance, etc. The amount of compound or compounds of the inventionthat is adequate to accomplish this is defined as a “therapeuticallyeffective dose.” The dosage schedule and amounts effective for this use,i.e., the “dosing regimen,” will depend upon a variety of factors,including the stage of the disease or condition, the severity of thedisease or condition, the general state of the patient's health, thepatient's physical status, age, pharmaceutical formulation andconcentration of active agent, and the like. In calculating the dosageregimen for a patient, the mode of administration also is taken intoconsideration. The dosage regimen must also take into consideration thepharmacokinetics, i.e., the pharmaceutical composition's rate ofabsorption, bioavailability, metabolism, clearance, and the like. See,e.g., the latest Remington's; Egleton (1997) “Bioavailability andtransport of peptides and peptide drugs into the brain” Peptides 18:14311439; Langer (1990) Science 249:1527-1533.

In therapeutic applications, compositions are administered to a patientsuffering from a cancer in an amount sufficient to at least partiallyarrest the disease and/or its complications. For example, in oneembodiment, a soluble pharmaceutical composition dosage for intravenous(IV) administration would be about 0.01 mg/hr to about 1.0 mg/hradministered over several hours (typically 1, 3, or 6 hours), which canbe repeated for weeks with intermittent cycles. Considerably higherdosages (e.g., ranging up to about 10 mg/ml) can be used, particularlywhen the drug is administered to a secluded site and not into the bloodstream, such as into a body cavity or into a lumen of an organ, e.g.,the cerebrospinal fluid (CSF).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Selective Abrogation of DNA-Damage-Induced Cell Cycle G2Checkpoint in G1 Checkpoint Defective Cells (Cancer Cells)

Chemicals and reagents. Bleomycin, adriamycin and colchicine werepurchased from Wako Pure Chemical Co. (Osaka, Japan). These chemicalswere dissolved in distilled H₂O at 10 mg/ml and stored at 4° C.Cisplatin was purchased from Nihon Kayaku Co. (Tokyo, Japan), dissolvedin distilled H₂O at 5 mg/ml, and stored at 4° C. Camptothecine (Campto)was purchased from Sigma Chemical Co. (St. Louis, Mo.). Propidium iodine(PI) was purchased from Sigma. Phytohemagglutinin was purchased fromLife Technologies, Inc. (Grand Island, N.Y.). Interleukin 2 (IL-2) waspurchased from Hemagen Diagnostics Inc.

Cell culture. A human T-cell leukemia-derived cell line, Jurkat, wascultured in RPMI 1640 media (Sigma) supplemented with 10% fetal calfserum (IBL: Immuno-Biological Laboratories, Gunma, Japan) at 37° C./5%CO₂. A human colon cancer-derived cell line, HCT116, was cultured inMcCoy's 5A Medium Modified (Gibco BRL) and supplemented with 10% fetalcalf serum at 37° C./5% CO₂. Normal human peripheral blood lymphocyteswere separated by Ficoll-Paque® (Pharmacia) and cultured in the presenceof 0.1 μg/ml PHA and 1 μg/ml IL-2.

Cell-cycle analysis. The cell cycle status of the cells treated withcompounds of the invention and/or bleomycin, adriamycin, colchicine, orcisplatin was analyzed by flow cytometry, essentially as described byKawabe (1997) Nature 385:454-458. Briefly, two million cells wereresuspended and incubated in 300 μl Krishan's solution (0.1% Sodiumcitrate, 50 μg/ml PI, 20 μg/ml RNase A and 0.5% NP-40) for 1 hr at 4° C.and analyzed by flow cytometry using a FACScan™ flow cytometer (BecktonDickinson, Mountain View, Calif.) with the CELLQuest™ program (BecktonDickinson).

CBDC402 Abolished Bleomycin-Induced Cell Cycle G2 Accumulation of JurkatCells.

Jurkat cells (a human T cell leukemia-derived cell line) were treatedwith bleomycin (40 μg/ml), or bleomycin plus compound CBDC402(4-bromo-benzoic acid p-tolyl ester) at various concentrations (0.2,0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 μg/ml) for 24 hrs. DNA oftreated Jurkat cells was stained with propidium iodine, and the cellcycle status of each cell was assessed by flow cytometry. As shown inFIG. 1, bleomycin treatment induced an accumulation of cells in cellcycle G2/M phase. CBDC402 treatment abolished the bleomycin-inducedaccumulation of G2/M cells in a dose-dependent manner.

M Phase Checkpoint in Jurkat Cells was not Abrogated by CBDC402Treatment.

Jurkat cells were treated with colchicine (5 ug/ml), and colchicine plusthe compound CBDC402 in various concentrations (0.2, 0.39, 0.78, 1.56,3.125, 6.25, 12.5, 25 and 50 ug/ml) for 24 hrs. DNA of treated Jurkatcells was stained with propidium iodine, and the cell cycle status ofeach cell was assessed by flow cytometry. As shown in FIG. 2, colchicinetreatment induced an accumulation of cells in G2/M phase cells, andCBDC402 did not abrogate this G2/M accumulation at any CBDC402concentration.

Various CBDC Compounds Abrogated the Cell Cycle G2 Checkpoint.

Jurkat cells were treated with bleomycin (40 ug/ml), and various CBDCcompounds at 0.2, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 ug/mland cultured for 24 hrs. CBDC compounds 004, 402, 403, 404, 405, 406,407, 408, 409, 410, and 411 were tested. Cells were harvested, DNA wasstained with propidium iodine, and the percentage of cells in G2/M phase(% G2/M) was determined by flow cytometry. FIG. 3 shows the % G2/M cellsthat were detected for each concentration of each CBDC compound,providing a dose-response curve for G2 checkpoint abrogation by variousCBDC compounds. CBDC402 showed the highest activity. All CBDC compoundstested abrogated G2 cell cycle checkpoint at the highest concentration(50 μg/ml). Structures of CBDC compounds tested in this experiment areshown in FIG. 4.

Additional CBDC compounds were tested for their G2 checkpoint abrogatingactivity as described above. As shown in FIG. 5, compounds were rankedas: highly active; moderately active; active; weakly active; andinactive. As shown in FIG. 6, the IC₅₀ of G2 checkpoint abrogation inJurkat cells was determined by dose-response studies carried out asdescribed above.

CBDC004 Abrogated Cell Cycle G2 Checkpoint Induced by VariousAnti-Cancer Agents.

HCT116 human colon carcinoma cells were treated with bleomycin (Bleo) at10 μg/ml, or adriamycin (ADR) at 1 μg/ml, or camptothecin (Campto) at 10μg/ml, or cisplatin (CDDP) at 2 μg/ml, and CBDC004 at 0, 2 μM, 10 μM, or50 μM, for 24 hrs. DNA was stained with propidium iodine, and the cellcycle status of each cell was assessed by flow cytometry. As shown inFIG. 7, each of these anti-cancer agents induced an accumulation ofcells at cell cycle G2/M, and co-incubation with CBDC004 reduced thenumber of cells at G2/M. This indicates that CBDC004 abrogated the G2checkpoint activated by bleomycin, adriamycin, camptothecin, orcisplatin.

Example 2 Sensitization of Cancer Cells to DNA-Damaging Treatment

The cytotoxic activity of combination treatments were determined byanalyzing subG1 population of HCT116 cells treated with bleomycin,adriamycine, canptothecine or cisplatin with or without CBDC004 at 2 μM,10 μM, or 50 μM. The subG1 population was determined by staining HCT116with Krishan's solution and analyzed by flow cytometer. The dead cellswere identified on the basis of DNA content, and the percentage of cellsin sub G1 (% subG1) was calculated. As shown in FIG. 8, bleomycin,adriamycine, canptothecine or cisplatin had a cytotoxic effect on HCT116cells, and CBDC004 increased the cytotoxic activities of theseanti-cancer agents in a dose dependent manner.

Example 3 CBDC402 Suppressed Xenograft Tumor Growth in SCID Mice

HCT-116 human colon carcinoma cells were implanted subcutaneously inSevere Combined Immunodeficiency (SCID) mice. Treatment was initiatedwhen the primary tumors reached the size of 0.1 cm³ (7 or 8 mm,designated as Day1).

Anti-cancer agent CPT-11 (CAMPTOSAR®, Irinotecan, a topoisomeraseinhibitor) and CBDC402 were administered by intraperitoneal injection inthe following treatment regimens: CPT-11 at 20 mg/kg once a week;CBDC402 at 20 mg/kg twice a week; CBDC402 at 40 mg/kg twice a week;CBDC402 at 20 mg/kg twice and week plus CPT-11 at 20 mg/kg once a week;and CBDC402 at 40 mg/kg twice a week plus CPT-11 at 20 mg/kg once aweek. DMSO was administered as a control treatment. Tumor sizes weremeasured using calipers three times a week, and the volume wascalculated using the formula; weight (mg)=[width (mm)²×length (mm)]/2.Mean tumor sizes for each treatment group (n=4) were plotted against thedays of treatment. As shown in FIG. 9, tumors in DMSO-treated controlmice continued to grow, CBDC402 alone (both concentrations) gave aslight reduction in tumor growth, CPT-11 alone reduced tumor growth, andcombinations of CBDC402 plus CPT-11 significantly reduced or inhibitedtumor growth.

Example 4 Colony Formation Analysis of Human Gastric Cancer Derived CellLine MK-45

MK-45 cells from a human gastric cancer derived cell line, were seededin 6-well plates at 1000 cells/well. After the overnight culture, thecells were treated 10 μM of various CBDC compounds, 10 μg/ml CPT-11, ora combination of CBDC compounds and CPT-11 for three (3) hours. The CBDCcompounds tested were: CBDC402, CBDC412, CBDC413, and CBDC418. Theculture medium was changed and cells were cultured for 14 days, afterwhich the colonies were fixed with methanol and stained with 0.1%crystal violet. A 3-hour treatment with 10 μM CBDC402 alone causedsignificant suppression of colony formation by MK-45 cells, whiletreatment with 10 μM CBDC412 caused a slight suppression of colonyformation, and 10 μM CBDC413, or CBDC418 did not have an appreciableeffect on colony formation. Treatment with 10 μg/ml CPT-11 causedsignificant suppression of colony formation, while addition of 10 μMCBDC402 appeared to augment the effect of 10 μg/ml CPT-11, resulting inalmost complete suppression of colony formation. The combination of 10μg/ml CPT-11 with 10 μM CBDC412 caused a slight suppression of colonyformation above that seen with CPT-11 alone, while combinations of 10μg/ml CPT-11 with 10 μM CBDC413, CBDC418.

Example 5 CBDC402 Specifically Abrogates the Cell Cycle G2 Checkpoint

Activated normal T cells and leukemic T cells (Jurkat cells) weretreated with agents to induce accumulation of cells at G2 or M phase,and the effect of CBDC402 on progression of cells through cell cyclecheckpoints was measured. After treatments, cells were harvested, DNAwas stained, and the cell cycle stage for each cell was determined byflow cytometry as described above. In one experiment, activated normal Tcells received no treatment (control), or were treated with bleomycin,CBDC402, or a combination of bleomycin and CBDC402. As shown in FIG. 10a, bleomycin treatment caused accumulation of a minor population ofcells at G2 phase, and CBDC402 abolished the bleomycin-induced increaseof activated normal T cells at G2 phase. In another experiment, leukemicT cells (Jurkat cells) received no treatment (control) or were treatedwith bleomycin, CBDC402, or a combination of bleomycin and CBDC402. Asshown in FIG. 10 b, bleomycin caused accumulation of a major populationof cells at G2 phase, and CBDC402 abolished the large bleomycin-inducedincrease of leukemic T cells (Jurkat cells) at G2 phase. In anotherexperiment, activated normal T cells received no treatment (control) orwere treated with colchicine or a combination of CBDC420 and colchicine.As shown in FIG. 10 c, colchicine caused an accumulation of cells in Mphase, and CBDC402 did not affect the colchicine-induced increase ofactivated normal T cells at M phase. These results indicated thespecificity of CBDC402 against G2 accumulation (and not M phasecheckpoint), and indicated the specificity of CBDC402 against cancercells.

Example 6 Synthesis of 4-bromo-benzoic acid 4-fluoro-phenyl ester

Ten ml of dioxane, 5 mmol (1.1 g) of 4-bromo-benzoic acid chlorine, and5 mmol (0.56 g) of 4-fluoro-phenol were added to 50 ml four-mouth-flasksequentially and dissolved at room temperature. Triethylamine dissolvedin dioxane was slowly dripped into this solution and the solution wasstirred for three hours at room temperature. The precipitated crystalswere filtered and extracted with benzene. The extracted solution waswashed with sodium hydrocarbonate several times, magnesium anhydrate wasadded, and the resulting solution was dried and filtered. The solutionwas distilled under low pressure and crystallized. The row crystals wereyellowish-white, weighing 1.37 g. A portion of this crystal (0.5 g) wasdissolved in benzene and purified with 100 g of silica gel (Wako gelC-300, Japan). The purified product was white, and the purity wasconfirmed to 99.93% by liquid chromatography (LC). The structure wasconfirmed by NMR as follows. ¹H NMR (400 MHz, DMSO-d6) δ 8.05 (2H, d,J=8.8 Hz), 7.83 (2H, d, J=8.8 Hz), 7.38-7.29 (4H, m) 13C NMR (100 MHz,DMSO-d6) δ 159.72 (C—F, d, J=240 Hz) 163.95 (C═O)

1-50. (canceled)
 51. A method for suppressing or killing a DNA-damagedcell, comprising administering an effective amount of at least onecompound that abrogates the cell cycle G2 checkpoint when administeredto a cell, wherein the compound has the following structure:

where either or both of benzene can be substituted with pyrazine,pyrimidine. piperazine, morpholine, cyclohexane, piperizine or pyridine;R1 is a halogen, bromine (Br), chlorine (Cl), fluorine (F), iodine (I),amino (NH₂), nitro (NO₂), hydroxy (OH), O-methyl (OCH₃), methyl (CH₃) orhydrogen (H); R2, R3, R4, R5 and R6 is bromine (Br), chlorine (Cl),fluorine (F), iodine (I), amino (NH₂), nitro (NO₂), methyl (CH₃),O-methyl (OCH₃), hydroxy (OH), CH(CH₃)₂, CHO, CHOCH₃, O(CH₂)nCH₃,OCO(C₆H₁₂)Cl, COOCH₃ or hydrogen; X1 is nitrogen (NH), oxygen (O) orsulfate (S); and X2 is oxygen (O) or sulfate (S).
 52. The method ofclaim 51, wherein the DNA-damaged cell has a DNA-damage-induced G2checkpoint.
 53. The method of claim 51, wherein the DNA-damaged cell hasan impaired G1 checkpoint
 54. The method of claim 51, wherein theDNA-damaged cell is a cancer cell.
 55. The method of claim 51, furthercomprising administering a DNA-damaging agent or treatment, whereinadministering the compound increases the sensitivity of the cell to theDNA-damaging agent or treatment.
 56. A method of selectively targetingDNA-damaged cells according to the method claim 51, comprisingadministering the compound to a population of cells comprisingDNA-damaged cells and normal cells, wherein abrogation of the G2checkpoint suppresses or kills DNA-damaged cells, and has little or nocytotoxic effect on normal cells.
 57. The method of claim 56, furthercomprising administering a DNA-damaging agent or treatment.
 58. Themethod of claim 56, wherein the DNA-damaged cells are cancer cells. 59.The method of claim 51, comprising administering the compound in vivo.60. The method of claim 51, comprising administering the compound exvivo.
 61. The method of claim 51, comprising administering the compoundin vitro.
 62. A method of treating a cell proliferative disorderaccording to claim 51, comprising administering the compound to asubject having a cell proliferative disorder characterized by aberrantor undesirable proliferation of at least one DNA-damaged cell.
 63. Themethod of claim 62, wherein the disorder is a neoplasm or cancer. 64.The method of claim 63, wherein the neoplasm or cancer is a tumor. 65.The method of claim 64, wherein the tumor is a solid tumor.
 66. Themethod of claim 64, wherein the tumor is a liquid tumor.
 67. The methodof claim 62, further comprising administering a DNA-damaging treatment.68. The method of claim 62, further comprising administering ananti-proliferative therapy.
 69. The method of claim 51, comprisingadministering at least one compound selected from: 4-chloro-benzoic acid4-methoxy-phenyl ester (CDBC004); 4-chloro-benzoic acid p-tolyl ester(CBDC401); 4-bromo-benzoic acid p-tolyl ester (CBDC402);3,4,5-trifluoro-benzoic acid p-toly 1 ester (CBDC403); 4-fluoro-benzoicacid 4-bromo-phenyl ester (CBDC404); 3,4-dichloro-benzoic acid p-tolylester (CBDC405); 2,4-dichloro-benzoic acid p-tolyl ester (CBDC406);4-fluoro-benzoic acid p-tolyl ester (CBDC407);2,3,4,5,6-pentafluoro-benzoic acid p-tolyl ester (CBDC408);4-chloro-benzoic acid 3,4-dimethyl-phenyl ester (CBDC409);4-chloro-benzoic acid 4-hydroxy-phenyl ester (CBDC410); 4-fluoro-benzoicacid 4-hydroxy-phenyl ester (CBDC411); 4-bromo-benzoic acid4-fluoro-phenyl ester (CBDC412); 4-bromo-benzoic acid4-trifluoromethyl-phenyl ester (CBDC413); 4-bromo-benzoic acid4-hydroxy-phenyl ester (CBDC414); 4-bromo-benzoic acid4-trifluoromethoxy-phenyl ester (CBDC415); 4-Bromo-benzoic acid6-methyl-pyridin-3-yl ester (CBDC418); 4-bromo-thiobenzoic acid0-p-tolyl ester (CBDC440); 4-bromo-dithiobenzoic acid p-tolyl ester(CBDC441); and 4-bromo-thiobenzoic acid S-p-tolyl ester (CBDC442). 70.The method of claim 51, comprising administering 4-chloro-benzoic acid4-methoxy-phenyl ester (CBDC004) having the structure:


71. The method of claim 51, comprising administering 4-chloro-benzoicacid p-tolyl ester (CBDC401) having the structure:


72. The method of claim 51, comprising administering 4-bromo-benzoicacid p-tolyl ester (CBDC402) having the structure:


73. The method of claim 51, comprising administering3,4,5-trifluoro-benzoic acid p-tolyl ester (CBDC402) having thestructure:


74. The method of claim 51, comprising administering 4-fluoro-benzoicacid 4-bromo-phenyl ester (CBDC404) having the structure:


75. The method of claim 51, comprising administering3,4-dichloro-benzoic acid p-tolyl ester (CBDC405) having the structure:


76. The method of claim 51, comprising administering2,4-dichloro-benzoic acid p-tolyl ester (CBDC406) having the structure:


77. The method of claim 51, comprising administering 4-fluoro-benzoicacid p-tolyl ester (CBDC407) having the structure:


78. The method of claim 51, comprising administering 2, 3, 4, 5,6-pentafluoro-benzoic acid p-tolyl ester (CBDC408) having the structure:


79. The method of claim 51, comprising administering 4-chloro-benzoicacid 3,4-dimethyl-phenyl ester (CBDC409) having the structure:


80. The method of claim 51, comprising administering 4-chloro-benzoicacid 4-hydroxy-phenyl ester (CBDC410) having the structure:


81. The method of claim 51, comprising administering 4-fluoro-benzoicacid 4-hydroxy-phenyl ester (CBDC411) having the structure:


82. The method of claim 51, comprising administering 4-bromo-benzoicacid 4-fluoro-phenyl ester (CBDC412) having the structure:


83. The method of claim 51, comprising administering 4-bromo-benzoicacid 4-trifluoromethyl-phenyl ester (CBDC413) having the structure:


84. The method of claim 51, comprising administering 4-bromo-benzoicacid 4-hydroxy-phenyl ester (CBDC414) having the structure:


85. The method of claim 51, comprising administering 4-bromo-benzoicacid 4-trifluoromethoxy-phenyl ester (CBDC415) having the structure:


86. The method of claim 51, comprising administering 4-bromo-benzoicacid 6-methyl-pyridin-3-yl ester (CBDC418) having the structure:


87. The method of claim 51, comprising administering 4-bromo-thiobenzoicacid 0-p-tolyl ester (CBDC440) having the structure:


88. The method of claim 51, comprising administering4-bromo-dithiobenzoic acid p-tolyl ester (CBDC441) having the structure:


89. The method of claim 51, comprising administering 4-bromo-thiobenzoicacid S-p-tolyl ester (CBDC442) having the structure:


90. A method of screening for non-peptide compounds that abrogate theDNA-damage-induced G2 checkpoint comprising: (a) providing a testcompound; (b) providing a population of cells; (c) administering to thepopulation of cells a DNA-damaging agent that causes accumulation ofG2/M phase cells; (d) administering the test compound to a first treatedportion of the population treated with the DNA-damaging agent; (e)measuring the number of cells in G2/M phase in the first treated portionof the population, wherein measuring the number of cells in G2/M phasein the population indicates the number of cells in G2 cell cycle arrest;(f) measuring the number of cells in G2/M phase in a second untreatedportion of the population treated with the DNA-damaging agent, whereinmeasuring the number of cells in G2/M phase in the population indicatesthe number of cells in G2 cell cycle arrest; and (g) comparing thenumber in (e) with the number in (f), wherein a lower number of cells inG2/M phase in step (e) indicates that the test compound abrogated theDNA-damage-induced G2 cell cycle checkpoint in the first treatedportion.
 91. The method of claim 90, wherein the test compound has thefollowing structure:

where either or both of benzene can be substituted with pyrazine,pyrimidine. piperazine, morpholine, cyclohexane, piperizine or pyridine;R1 is a halogen, bromine (Br), chlorine (Cl), fluorine (F), iodine (I),amino (NH₂), nitro (NO₂), hydroxy (OH), O-methyl (OCH₃), methyl (CH₃) orhydrogen (H); R2, R3, R4, R5 and R6 is bromine (Br), chlorine (Cl),fluorine (F), iodine (I), amino (NH₂), nitro (NO₂), methyl (CH₃),O-methyl (OCH₃), hydroxy (OH), CH(CH₃)₂, CHO, CHOCH₃, O(CH₂)_(n)CH₃,OCO(C₆H₁₂)Cl, COOCH₃ or hydrogen; X1 is nitrogen (NH), oxygen (O) orsulfate (S); and X2 is oxygen (O) or sulfate (S).